Methods of inducing regulated pancreatic hormone production in non-pancreatic islet tissues

ABSTRACT

Disclosed are methods and pharmaceutical compositions for inducing pancreatic hormone production.

RELATED APPLICATIONS

This application claims priority to U.S. Ser. No. 60/137,143 filed Jun.1, 1999 and U.S. Ser. No. 60/198,532 filed Apr. 19, 2000. The contentsof these applications are incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

This invention relates generally to methods of inducing a pancreaticendocrine phenotype and function including pancreatic hormone productionin a non-endocrine tissue and in particular to methods andpharmaceutical compositions for treating endocrine related disorders.

BACKGROUND OF THE INVENTION

The endocrine pancreas consists primarily of islet cells that synthesizeand secrete the peptide hormone glucagon, insulin, somatostatin andpancreatic polypeptide. Insulin gene expression is restricted topancreatic islet 6-cells of the mammalian pancreas through control pusmechanisms mediated in part by specific transcription factors. In othercells the insulin, other pancreatic hormones and specific peptidasesgenes are trancriptionally silent. The homeodomain protein PDX-1(Pancreatic and Duodenal Homeobox gene-1, also known-as IDX-1, IPF-1,STF-1 or IUF-1) plays a central role in regulating pancreatic isletdevelopment and function. PDX-1 is either directly or indirectlyinvolved in islet-cell-specific expression of various genes such as forexample insulin, glucagon somatostatin, proinsulin convertase 1/3 (PC1/3), GLUT-2 and glucokinase. Additionally, PDX-1 mediates insulin genetranscription in response to glucose.

SUMMARY OF THE INVENTION

The invention is based in part on the discovery that ectopic expressionof pancreatic and duodenal homobox gene 1 (PDX-1) in liver induces theexpression of the silent pancreatic hormone genes and the processingmachinery, which converts the prohormones into mature biologicallyactive hormones.

The invention provides methods of inducing pancreatic hormone, e.g.,insulin, glucagon and somatostatin levels in a subject. In one aspect,the method includes administering to a subject in need thereof acompound which increases PDX expression or activity in an amountsufficient to induce pancreatic hormone production in the subject. Inanother aspect, the method includes providing a cell capable ofexpressing a pancreatic hormone, contacting the cell with a compoundwhich increases PDX expression or activity and introducing the cell intoa subject, thereby inducing pancreatic hormone production in thesubject. Also provided in the invention is a method of treating apancreatic-related disorder, e.g.,

diabetes in a subject. The method includes administering to a subject atherapeutically effective amount of a compound which increases PDXexpression.

In another aspect the invention provides a method of inducing apancreatic islet gene expression profile in a subject. The methodincludes administering to a subject in need thereof a compound whichincreases PDX expression or activity in an amount sufficient to inducepancreatic islet gene expression.

In yet a further aspect of the invention is a method inducing orenhancing a pancreatic islet cell phenotype in a cell. The methodincludes contacting a cell with compound which increases PDX expressionor activity in an amount sufficient to induce or enhance pancreaticislet cell phenotype in said cell.

Also included are pharmaceutical composition that includes a compoundwhich increases PDX expression and a pharmaceutically acceptablecarrier.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration demonstrating detection of mRNA in Balb/c miceliver tissue for mouse insulin I (mI-1), mouse insulin II (mI-2), humaninsulin, PDX-1 and β-actin after adenovirus treatment as determined byRT-PCR. Lane 1: no DNA (negative control for PCR); lanes 2-6: liversfrom AdCMV-PDX-1 treated mice lanes 7, 8: livers fromAdCMV-PDX-1+AdRIP-1-hIns-treated mice; lanes 9-11: livers from controlAdCMV-β-gal+AdRIP-1-hIns-treated mice; lanes 12, 13: livers fromAdCMV-hIns-treated mice; lane 14: normal mouse pancreas.

FIG. 2 is an illustration of the HPLC elution profiles of insulinrelated peptides extracted from murine tissue. Panel A shows the profilefrom the pancreas of a PDX-1 treated mouse. Panel B shows the profilefrom the liver of a PDX-1 treated mouse.

FIG. 3 is an illustration demonstrating detection of mRNA for PDX-1,somatostatin in Somato), proinsulin convertase PC1/3 (PC1/3), glucagon(Glucg) and β-actin determined by RT-PCR: Total RNA extracted from PDX-1and control treated mice was reverse-transcribed using a PC1/3 specificprimer lanes 1-3: mice treated by AdCMV-PDX-1; lanes 4-5: mice treatedby AdCMV-β-gal; lane 6: pancreas; lane 7: no cDNA, (control for PCR).

FIG. 4 is an illustration demonstrating ectopic PDX-1 expression in micelivers ameliorates STZ induced hyperglycemia: C57BL/6 males at 12-13weeks were treated by 220 mg/kg STZ in citrate-buffer. 36-48 hour afterSTZ treatment mice were injected by AdCMVPDX-1 (n=15 mice), or ascontrol by AdCMVβ-gal (n=22, however, 12 died 3-5 days after STZtreatment, additional 3 mice died 6-7 days after STZ treatment). Nomortality occurred upon AdCMVPDX-1 treatment. Each treatment includedsystemic injection of 2×10⁹ PFU (plaque forming units) of recombinantadenovirus in 200 μl saline. Glucose levels were determined in bloodsamples drawn from the ocular vein.

FIG. 5 is an illustration demonstrating ectopic PDX expression in maturehepatocytes in culture activates insulin promoter (rat insulin-1promoter), co-delivered to the same cells by AdRIPhIns. Human insulin isdetected as in FIG. 1. Lane 1: cells treated by AdCMV PDX-1+AdRIP hIns,lane 2: AdCMVβ-galactosidase+AdRIP hIns, lane 3: Control.

FIG. 6 is an illustration demonstrating the induction of Insulin 1 andSomatostatin gene expression in primary monolayer cultures of fetalFisher rat (E14) hepatocytes. Fetal hepatocytes were isolated fromFisher 344 rat embryos at day 14 of gestation, and plated on collagencovered tissue culture dishes. Cells were infected by AdCMVPDX-1 at 2-5MOI (multipiplicity of infection=number of viral particles per cell).Total RNA was extracted from the culture 4 days after viral treatmentand was analyzed for somatostatin gene expression by RT-PCR. RNA wasreversed transcribed as in FIG. 1 using oligo (dT)₁₅ primers andamplification by PCR was in performed using primers and conditions aselaborated in Table 1. Lanes 1-3: samples from cells treated by PDX-1,lanes 4-6: untreated samples (control) lane 7: no DNA, PCR products wereresolved on 1.7% agarose gel electrophoresis.

FIG. 7 is an illustration demonstrating the Effect of GLUT2 and GKoverexpression on the glucose regulation of PDX-1 binding to A3/A4 siteon the insulin promoter. RIN-38 cells of intermediate passage werestudied untreated (lane 1), treated with AdCMV-GLUT2 or AdCMV-GK (lanes3,4), or with control AdCMVCAT (lane 5). 48 hours after viraltreatments, nuclear extracts were prepared and EMSA analysis wasperformed using the A3/A4 sequences as probes. Last two lanes: 1 μl ofanti-PDX-1 antibody (a gift from C. Wright) was added to the nuclearextract (+) and blocked complex formation. Pre-immune serum was added tothe nuclear extract (−) used to identify that PDX-1 is indeed theprotein bound to the given portion of the insulin promoter.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based in part on the discovery that ectopic expressionof pancreatic and duodenal homobox gene 1 (PDX-1) in liver induces apancreatic islet cell phenotype in liver cells and results in theexpression, production and processing of pancreatic hormones. PDX-1 isalso known as IDX-1, IPF-1, STF-1 and IUF-1, all of which arecollectively referred to herein as “PDX”. Additionally, the inventionprovides methods and pharmaceutical compositions for treating pancreaticdisorders.

In its various aspects and embodiments, the a invention includesadministering to a subject or contacting a cell with a compound thatincreases PDX expression or activity. The compound can be, e.g., (i) aPDX polypeptide; (ii) a nucleic acid encoding a PDX polypeptide; (iii) anucleic acid that increases expression of a nucleic acid that encodes aPDX polypeptide and, and derivatives, fragments, analogs and homologsthereof. A nucleic acid that increase expression of a nucleic acid thatencodes a PDX polypeptide includes, e.g., promoters, enhancers. Thenucleic acid can be either endogenous or exogenous.

As used herein, the term “nucleic acid” is intended to include DNAmolecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA),analogs of the DNA or RNA generated using nucleotide analogs, andderivatives, fragments and homologs thereof. The nucleic acid moleculecan be single-stranded or double-stranded. Preferably, the nucleic acidis a DNA. A nucleic acid that increase expression of a nucleic acid thatencodes a PDX polypeptide includes, e.g., promoters, enhancers. Thenucleic acid can be either endogenous or exogenous.

Suitable sources of nucleic acids encoding PDX include for example thehuman PDX nucleic acid (and the encoded protein sequences) available asGenBank Accession Nos. U35632 and AAA88820, respectively. Other sourcesinclude rat PDX nucleic acid and protein sequences are shown in GenBankAccession No. U35632 and AAA18355, respectively, and are incorporatedherein by reference in their entirety. An addition source includezebrafish PDX nucleic acid and protein sequences are shown in GenBankAccession No. AF036325 and AAC41260, respectively, and are incorporatedherein by reference in their entirety.

The compound can be administered to the subject either directly (i.e.,the subject is directly exposed to the nucleic acid or nucleicacid-containing vector) or indirectly (i.e., cells are first transformedwith the nucleic acid in vitro, then transplanted into the subject). Forexample, in one embodiment mammalian cells are isolated from a subjectand the PDX nucleic acid introduced into the isolated cells in vitro.The cells are reintroduced into a suitable mammalian subject.Preferably, the cell is introduced into an autologous subject. Theroutes of administration of the compound can include e.g., parenteral,intravenous, intradermal, subcutaneous, oral (e.g., inhalation),transdermal (topical), transmucosal, and rectal administration. In oneembodiment the compound is administered intravenous. Preferably, thecompound is implanted under the kidney capsule or injected into theportal vein.

The cell can be any cell that is capable of producing pancreatichormones, e.g., muscle, spleen, kidney, blood, skin, pancreas, or liver.In one embodiment the cell is capable of functioning as a pancreaticislet cell, i.e., store, process and secrete pancreatic hormones,preferably insulin upon an extracellular trigger. In another embodimentthe cell is a hepatocyte, i.e., a liver cell. In additional embodimentsthe cell is tutipont or pluripotent. In alternative embodiments the cellis a hemopoietic stem cell, embryonic stem cell or preferably a hepaticstem cell.

The subject is preferably a mammal. The mammal can be, e.g., a human,non-human primate, mouse, rat, dog, cat, horse, or cow.

Methods of Inducing Pancreatic Hormone Production

In various aspects, the invention provides methods of inducingpancreatic hormone production in a subject. For example, the method caninclude administering to a subject a compound that increases PDXexpression or activity in an amount sufficient to induce pancreatichormone production.

In another aspect, the method includes providing a cell from a subject,contacting the cell with a compound which increases PDX expression in anamount sufficient to increase pancreatic hormone production andintroducing the cell into a subject. In one embodiment pancreatichormone production occurs in-vitro and in-vivo, upon introducing thecell into the subject. In an alternative embodiment, pancreatic hormoneproduction occurs in-vivo upon introducing the cell in the subject.

The pancreatic hormone can be e.g., insulin, glucogon, somatostatin orislet amyloid polypeptide (IAPP). Insulin can be hepatic insulin orserum insulin. In another embodiment the pancreatic hormone is hepaticinsulin. In an alternative embodiment the pancreatic hormone is seruminsulin (i.e., a fully processed form of insulin capable of promoting,e.g., glucose utilization, carbohydrate, fat and protein metabolism).

In some embodiments the pancreatic hormone is in the “prohormone” form.In other embodiments the pancreatic hormone is in the fully processedbiologically active form of the hormone. In other embodiments thepancreatic hormone is under regulatory control i.e., secretion of thehormone is under nutritional and hormonal control similar toendogenously produced pancreatic hormones. For example, in one aspect ofthe invention the hormone is under the regulatory control of glucose.

The cell population that is exposed to, i.e., contacted with, thecompound can be any number of cells, i.e., one or more cells, and can beprovided in vitro, in vivo, or ex vivo.

Methods of Treating or Preventing Pancreatic Related Disorders

Also included in the invention is a method of treating, i.e., preventingor delaying the onset of pancreatic related disorders in a subject. Invarious aspects the method includes administering to the subject acompound which modulates the PDX expression or activity. “Modulates” ismeant to include increase or decrease PDX expression or activity.Preferably, modulation results in alteration of the expression oractivity of PDX in a subject to a level similar or identical to asubject not suffering from the pancreatic disorder. In other aspects themethod includes administering to the subject a compound which induces anon-pancreatic cell with pancreatic islet cell function, e.g., capableof expressing insulin, somatostatin or glucagon. In one embodiment thecompound modulates PDX expression or activity.

The pancreatic disorder can be any disorder associated with thepancreas. For example, the method may be useful in treating pancreatichormone insufficiencies, (e.g., diabetes), insulinomas, andhyperglycemia. Essentially, any disorder, which is etiologically linkedto PDX activity, would be considered susceptible to treatment.

The herein-described PDX modulating compound when used therapeuticallyare referred to herein as “Therapeutics”. Methods of administration ofTherapeutics include, but are not limited to, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes. The Therapeutics of the present invention maybe administered by any convenient route, for example by infusion orbolus injection, by absorption through epithelial or mucocutaneouslinings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and maybe administered together with other biologically-active agents.Administration can be systemic or local. In addition, it may beadvantageous to administer the Therapeutic into the central nervoussystem by any suitable route, including intraventricular and intrathecalinjection. Intraventricular injection may be facilitated by anintraventricular catheter attached to a reservoir (e.g. an Ommayareservoir). Pulmonary administration may also be employed by use of aninhaler or nebulizer, and formulation with an aerosolizing agent. It mayalso be desirable to administer the Therapeutic locally to the area inneed of treatment; this may be achieved by, for example, and not by wayof limitation, local infusion during surgery, topical application, byinjection, by means of a catheter, by means of a suppository, or bymeans of an implant. Various delivery systems are known and can be usedto administer a Therapeutic of the present invention including, e.g.:(i) encapsulation in liposomes, microparticles, microcapsules; (ii)recombinant cells capable of expressing the Therapeutic; (iii)receptor-mediated endocytosis (See, e.g., Wu and Wu, 1987. J Biol Chem262:4429-4432); (iv) construction of a Therapeutic nucleic acid as partof a retroviral, adenoviral or other vector, and the like. In oneembodiment of the present invention, the Therapeutic may be delivered ina vesicle, in particular a liposome. In a liposome, the protein of thepresent invention is combined, in addition to other pharmaceuticallyacceptable carriers, with amphipathic agents such as lipids which existin aggregated form as micelles, insoluble monolayers, liquid crystals,or lamellar layers in aqueous solution. Suitable lipids for liposomalformulation include, without limitation, monoglycerides, diglycerides,sulfatides, lysolecithin, phospholipids, saponin, bile acids, and thelike. Preparation of such liposomal formulations is within the level ofskill in the art, as disclosed, for example, in U.S. Pat. No. 4,837,028;and U.S. Pat. No. 4,737,323, all of which are incorporated herein byreference. In yet another embodiment, the Therapeutic can be deliveredin a controlled release system including, e.g.: a delivery pump (See,e.g., Saudek, et al., 1989. New Engl J Med 321:574 and a semi-permeablepolymeric material (See, e.g., Howard, et al., 1989. J Neurosurg71:105).

Additionally, the controlled release system can be placed in proximityof the therapeutic target (e.g., the brain), thus requiring only afraction of the systemic dose. See, e.g., Goodson, In: MedicalApplications of Controlled Release 1984. (CRC Press, Bocca Raton, Fla.).

In a specific embodiment of the present invention, where the Therapeuticis a nucleic acid encoding a protein, the Therapeutic nucleic acid maybe administered in vivo to promote expression of its encoded protein, byconstructing it as part of an appropriate nucleic acid expression vectorand administering it so that it becomes intracellular (e.g., by use of aretroviral vector, by direct injection, by use of microparticlebombardment, by coating with lipids or cell-surface receptors ortransfecting agents, or by administering it in linkage to ahomeobox-like peptide which is known to enter the nucleus (See, e.g.,Joliot, et al., 1991. Proc Natl Acad Sci USA 88:1864-1868), and thelike. Alternatively, a nucleic acid Therapeutic can be introducedintracellularly and incorporated within host cell DNA for expression, byhomologous recombination or remain episomal.

As used herein, the term “therapeutically effective amount” means thetotal amount of each active component of the pharmaceutical compositionor method that is sufficient to show a meaningful patient benefit, i.e.,treatment, healing, prevention or amelioration of the relevant medicalcondition, or an increase in rate of treatment, healing, prevention oramelioration of such conditions. When applied to an individual activeingredient, administered alone, the term refers to that ingredientalone. When applied to a combination, the term refers to combinedamounts of by the active ingredients that result in the therapeuticeffect, whether administered in combination, serially or simultaneously.

The amount of the Therapeutic of the invention which will be effectivein the treatment of a particular disorder or condition will depend onthe nature of the disorder or condition, and may be determined bystandard clinical techniques by those of average skill within the art.In addition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theoverall seriousness of the disease or disorder, and should be decidedaccording to the judgment of the practitioner and each patient'scircumstances. Ultimately, the attending physician will decide theamount of protein of the present invention with which to treat eachindividual patient. Initially, the attending physician will administerlow doses of protein of the present invention and observe the patient'sresponse. Larger doses of protein of the present invention may beadministered until the optimal therapeutic effect is obtained for thepatient, and at that point the dosage is not increased further. However,suitable dosage ranges for intravenous administration of theTherapeutics of the present invention are generally about 20-500micrograms (μg) of active compound per kilogram (Kg) body weight.Suitable dosage ranges for intranasal administration are generally about0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems. Suppositories generally contain active ingredient inthe range of 0.5% to 10% by weight; oral formulations preferably contain10% to 95% active ingredient The duration of intravenous therapy usingthe Therapeutic of the present invention will vary, depending on theseverity of the disease being treated and the condition and potentialidiosyncratic response of each individual patient. It is contemplatedthat the duration of each in application of the protein of the presentinvention will be in the range of 12 to 24 hours of continuousintravenous administration. Ultimately the attending physician willdecide on the appropriate duration of intravenous therapy using thepharmaceutical composition of the present invention.

Cells may also be cultured ex vivo in the presence of therapeutic agentsor proteins of the present invention in order to proliferate or toproduce a desired effect on or activity in such cells. Treated cells canthen be introduced in vivo for therapeutic purposes.

Methods of Inducing Islet Cell Phenotype and Function

The invention also includes a method of inducing or enhancing a one ormore pancreatic islet cell phenotypes in a cell. In one embodiment thepancreatic cell phenotype is induced in a non-islet cell type. Themethod includes contacting a cell with a compound that modulates isletcell specific transcription factors in an amount sufficient to induce orenhance the pancreatic islet cell phenotype, e.g., beta, alpha and deltaislet cells. Preferably, the compound increases PDX expression,production or activity. Preferably the method induces a pancreatic isletβ-cell phenotype.

By “ pancreatic islet cell phenotype” is meant that the cell displayingone or more characteristics typical of pancreatic islet cell i.e.hormone production, processing, storage in secretory granules,nutritionally and hormonally regulated secretion or characteristic isletcell gene expression profile. The pancreatic islet cell phenotype can bedetermined for example by measuring pancreatic hormone production, e.g.,insulin, somatostatin or glucagon. Hormone production can be determinedby methods known in the art, e.g. immunoassay, western blot, receptorbinding assays or functionally by the ability to amelioratehyperglycemia implantation in a diabetic host.

The cell can be any cell that is capable of expressing a pancreaticislet cell phenotype, e.g., muscle, spleen, kidney, skin, pancreas, orliver. In one embodiment the cell is capable of functioning as apancreatic islet cell, i.e., store, process and secrete pancreatichormones, preferably insulin upon an extracellular trigger. In anotherembodiment the cell is a hepatocyte, i.e., a liver cell. In additionalembodiments the cell is tutipont or pluripotent. In alternativeembodiments the cell is a hematopoietic stem cell, embryonic stem cellor preferably a hepatic stem cell.

The cell population that is exposed to, i.e., contacted with, thecompound can be any number of cells, i.e., one or more cells, and can beprovided in vitro, in vivo, or ex vivo.

Methods of Inducing a Pancreatic Islet Gene Expression Profile

The invention also includes a method of inducing or enhancing apancreatic islet gene expression profile in a subject or a cell. By“pancreatic gene expression profile” is meant to include one or moregenes that are normally transcriptionally silent in non-endocrinetissues, e.g., PC1/3, insulin, glucagon or somatostatin. The methodincludes administering to a subject a compound that increases PDXexpression or activity in an amount sufficient to induce a pancreaticislet or endocrine gene expression profile. In one embodiment the methodinduces PC1/3 gene expression in a subject.

Induction of the pancreatic gene expression profile can be detectedusing techniques well known to one of ordinary skill in the art. Forexample, pancreatic hormone RNA sequences can be detected in, e.g.,northern blot hybridization analyses, amplification-based detectionmethods such as reverse-transcription based polymerase chain reaction orsytemic detection by microarray chip analysis. Alternatively, expressioncan be also measured at the protein level, i.e., by measuring the levelsof polypeptides encoded by the gene. In a specific embodiment PC1/3 geneor protein expression can be determined by its activity in processingprohormones to their active mature form. Such methods are well known inthe art and include, e.g., immunoassays based on antibodies to proteinsencoded by the genes, or HPLC of the processed prohormones.

Methods of Identifying Genes the Expression of Which is Modulated by PDX

The invention also includes a method of identifying nucleic acids theexpression of which is modulated by PDX. The method includes measuringthe expression of one or more nucleic acids in a test cell populationexposed to a compound that modulates PDX activity or expression.Expression of the nucleic acid sequences in the test cell population isthen compared to the expression of the nucleic acid sequences in areference cell population, which is a cell population that has not beenexposed to the compound, or, in some embodiments, a cell populationexposed the compound. Comparison can be performed on test and referencesamples measured concurrently or at temporally distinct times. Anexample of the latter is the use of compiled expression information,e.g., a sequence database, which assembles information about expressionlevels of known sequences following administration of various agents,For example, alteration of expression levels following administration ofcompound can be compared to the expression changes observed in thenucleic acid sequences following administration of a control agent, sucha PDX nucleic acid.

An alteration in expression of the nucleic acid sequence in the testcell population compared to the expression of the nucleic acid sequencein the reference cell population that has not been exposed to thecompound indicates expression of the nucleic acid is modulated by PDX.The test cell can be taken from any tissue capable of being modulated byPDX, e.g., pancreas, liver, spleen, or kidney. In one embodiment thecell is from a non-endocrine tissue. Preferably, the cell is livertissue.

Preferably, cells in the reference cell population are derived from atissue type as similar as possible to test cell, e.g., liver tissue. Insome embodiments, the control cell is derived from the same subject asthe test cell, e.g., from a region proximal to the region of origin ofthe test cell. In other embodiments, the control cell population isderived from a database of molecular information derived from cells forwhich the assayed parameter or condition is known.

Expression of the nucleic acids can be measured at the RNA level usingany method known in the art. For example, northern hybridizationanalysis using probes which specifically recognize one or more of thesesequences can be used to determine gene expression. Alternatively,expression can be measured using reverse-transcription-based PCR assays.Expression can be also measured at the protein level, i.e., by measuringthe levels of polypeptides encoded by the gene products. Such methodsare well known in the art and include, e.g., immunoassays based onantibodies to proteins encoded by the genes.

When alterations in gene expression are associated with geneamplification or deletion, sequence comparisons in test and referencepopulations can be made by comparing relative amounts of the examinedDNA sequences in the test and reference cell populations.

The invention also includes PDX modulated nucleic acids identifiedaccording to this screening method, and a pharmaceutical compositioncomprising the PDX modulated nucleic acids so identified.

PDX Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a PDX protein, orderivatives, fragments, analogs or homologs thereof. As used herein, theterm “vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. One type of vector isa “plasmid”, which refers to a linear or circular double stranded DNAloop into which additional DNA segments can be ligated. Another type ofvector is a viral vector, wherein additional DNA segments can be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)are integrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “expression vectors”. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.In the present specification, “plasmid” and “vector” can be usedinterchangeably as the plasmid is the most commonly used form of vector.However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions. Additionally, some viral vectors are capable oftargeting a particular cells type either specifically ornon-specifically.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, that is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerthat allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to includes promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; GENE EXPRESSION TECHNOLOGY: METHODSIN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those that direct constitutive expression of anucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., PDX proteins, mutant forms ofPDX, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of PDX in prokaryotic or eukaryotic cells. For example, PDXcan be expressed in bacterial cells such as E. coli, insect cells (usingbaculovirus expression vectors) yeast cells or mammalian cells. Suitablehost cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).Alternatively, the recombinant expression vector can be transcribed andtranslated in vitro, for example using T7 promoter regulatory sequencesand T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: (1) to increase expression ofrecombinant protein; (2) to increase the solubility of the recombinantprotein; and (3) to aid in the purification of the recombinant proteinby acting as a ligand in affinity purification. Often, in fusionexpression vectors, a proteolytic cleavage site is introduced at thejunction of the fusion moiety and the recombinant protein to enableseparation of the recombinant protein from the fusion moiety subsequentto purification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amrann el al., (1988) Gene 69:301-315) and pET 11d(Studier el al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185,Academic Press, San Diego, Calif. (1990) 60-89).

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein. See, Gottesman, GENEEXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, SanDiego, Calif. (1990) 119-128. Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the PDX expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari, et al., (1987) EMBO J 6:229-234), pMFa (Kujanand Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987)Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), andpicZ (InVitrogen Corp, San Diego, Calif.).

Alternatively, PDX can be expressed in insect cells using baculovirusexpression vectors. Baculovirus vectors available for expression ofproteins in cultured insect cells (e.g., SF9 cells) include the pAcseries (Smith et al. (1983) Mol Cell Biol 3:2156-2165) and the pVLseries (Lucklow and Summers (1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840)and pMT2PC (Kaufinan et al. (1987) EMBO J 6: 187-195). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells. See, e.g., Chapters 16 and 17 ofSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv Immunol 43:235-275), in particular promoters of T cellreceptors (Winoto and Baltimore (1989) EMBO J 8:729-733) andimmunoglobulins (Baneiji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477),pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, e.g., themurine hox promoters (Kessel and Gruss (1990) Science 249:374-379) andthe α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner that allows forexpression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to PDX in mRNA. Regulatory sequences operativelylinked to a nucleic acid cloned in the antisense orientation can bechosen that direct the continuous expression of the antisense RNAmolecule in a variety of cell types, for instance viral promoters and/orenhancers, or regulatory sequences can be chosen that directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub elal., “Antisense RNA as a molecular tool for genetic analysis,”Reviews-Trends in Genetics, Vol. 1(1) 1986.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but also to the progeny or potential progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein. Additionally, hostcells could be modulated once expressing PDX, and may either maintain orloose original characteristics.

A host cell can be any prokaryotic or eukaryotic cell. For example, PDXprotein can be expressed in bacterial cells such as E. coli, insectcells, yeast or mammalian cells (such as Chinese hamster ovary cells(CHO) or COS cells). Alternatively, a host cell can be a prematuremammalian cell, i.e., pluripotent stem cell. A host cell can also bederived from other human tissue. Other suitable host cells are known tothose skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation,transduction, infection or transfectiontechniques. As used herein, the terms At “transformation”“transduction”, “infection” and “transfection” are intended to refer toa variety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. In addition transfection can bemediated by a transfection agent. By “transfection agent” is meant toinclude any compound that mediates incorporation of DNA in the hostcell, e.g., liposome. Suitable methods for transforming or transfectinghost cells can be found in Sambrook, el al. (MOLECULAR CLONING: ALABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and otherlaboratory manuals.

Transfection may be “stable” ( i.e. intergration of the foreign DNA intothe host genome) or “transient” (i.e., DNA is episomally expressed inthe host cells).

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome theremainder of the DNA remains episomal In order to identify and selectthese integrants, a gene that encodes a selectable marker (e.g.,resistance to antibiotics) is generally introduced into the host cellsalong with the gene of interest. Various selectable markers includethose that confer resistance to drugs, such as G418, hygromycin andmethotrexate. Nucleic acid encoding a selectable marker can beintroduced into a host cell on the same vector as that encoding PDX orcan be introduced on a separate vector. Cells stably transfected withthe introduced nucleic acid can be identified by drug selection (e.g.,cells that have incorporated the selectable marker gene will survive,while the other cells die). In another embodiment the cells modulated byPDX or the transfected cells are identified by the induction ofexpression of a endogeneous reporter gene. In a specific embodiment, thepromotor is the insulin promoter driving the expression of greenflourescent protein (GFP).

In one embodiment the PDX nucleic acid is present in a viral vector. Inanother embodiment the PDX nucleic acid is encapsulated in a virus. Insome embodiments the virus preferably infects pluripotent cells ofvarious tissue type, e.g. hematopoietic stem, cells, neuronal stemcells, hepatic stem cells or embroyonic stem cells, preferably the virusis hepatropic. By “hepatotropic” it is meant that the virus has thecapacity to preferably target the cells of the liver girt eitherspecifically or non-specifically. In further embodiments the virus is amodulated hepatitis virus, SV40, or Epstein-Bar virus. In yet anotherembodiment, the virus is an adenovirus.

Gene Therapy

In one aspect of the invention a nucleic acid or nucleic acids encodinga PDX polypeptide, or functional derivatives thereof, are administeredby way of gene therapy. Gene therapy refers to therapy that is performedby the administration of a specific nucleic acid to a subject. In thisaspect of the invention, the nucleic acid produces its encodedpeptide(s), which then serve to exert a therapeutic effect by modulatingfunction of an aforementioned disease or disorder. e.g., diabetes. Anyof the methodologies relating to gene therapy available within the artmay be used in the practice of the present invention. See e.g.,Goldspiel, et at., 1993. Clin Pharm 12:488-505.

In a preferred embodiment, the therapeutic comprises a nucleic acid thatis part of an expression vector expressing any one or more of theaforementioned PDX polypeptides, or fragments, derivatives or analogsthereof, within a suitable host. In a specific embodiment, such anucleic acid possesses a promoter that is operably-1inked to codingregion(s) of a PDX polypeptide. The promoter may be inducible orconstitutive, and, optionally, tissue-specific. The promoter may be,e.g., viral or mammalina in origin. In another specific embodiment, anucleic acid molecule is used in which coding sequences (and any otherdesired sequences) are flanked by regions that promote homologousrecombination at a desired site within the genome, thus providing forintra-chromosomal expression of nucleic acids. See e.g., Koller andSmithies, 1989. Proc Natl Acad Sci USA 86: 8932-8935. In yet anotherembodiment the nucleic acid that is delivered remains episomal andinduces an endogenous and otherwise silent gene.

Delivery of the therapeutic nucleic acid into a patient may be eitherdirect (i.e., the patient is directly exposed to the nucleic acid ornucleic acid-containing vector) or indirect (i.e., cells are firstcontacted with the nucleic acid in vitro, then transplanted into thepatient). These two approaches are known, respectively, as in vivo or exvivo gene therapy. In a specific embodiment of the present invention, anucleic acid is directly administered in vivo, where it is expressed toproduce the encoded product. This may be accomplished by any of numerousmethods known in the art including, but not limited to, constructingsaid nucleic acid as part of an appropriate nucleic acid expressionvector and administering the same in a manner such that it becomesintracellular (e.g., by infection using a defective or attenuatedretroviral or other viral vector; see U.S. Pat. No. 4,980,286); directlyinjecting naked DNA; using microparticle bombardment (e.g., a “GeneGun®; Biolistic, DuPont); coating said nucleic acids with lipids; usingassociated cell-surface receptors/transfecting agents; encapsulating inliposomes, microparticles, or microcapsules; administering it in linkageto a peptide that is known to enter the nucleus; or by administering itin linkage to a ligand predisposed to receptor-mediated endocytosis(see, e.g., Wu and Wu, 1987. J Biol Chem 262: 4429-4432), which can beused to “target” cell types that specifically express the receptors ofinterest, etc.

An additional approach to gene therapy in the practice of the presentinvention involves transferring a gene into cells in in vitro tissueculture by such methods as electroporation, lipofection, calciumphosphate-mediated transfection, viral infection, or the like.Generally, the methodology of transfer includes the concomitant transferof a selectable marker to the cells. The cells are then placed underselection pressure (e.g., antibiotic resistance) so as to facilitate theisolation of those cells that have taken up, and are expressing, thetransferred gene. Those cells are then delivered to a patient. In aspecific embodiment, prior to the in vivo administration of theresulting recombinant cell, the nucleic acid is introduced into a cellby any method known within the art including, but not limited to:transfection, electroporation, microinjection, infection with a viral orbacteriophage vector containing the nucleic acid sequences of interest,cell fusion, chromosome-mediated gene transfer, microcell-mediated genetransfer, spheroplast fusion, and similar methodologies that ensure thatthe necessary developmental and physiological functions of the recipientcells are not disrupted by the transfer. See e.g., Loeffler and Behr,1993. Meth Enzymol 217: 599-618. The chosen technique should provide forthe stable transfer of the nucleic acid to the cell, such that thenucleic acid is expressible by the cell. Preferably, said transferrednucleic acid is heritable and expressible by the cell progeny. In analterantive embodiment, the transferred nucleic acid remains episomaland induces the expression of the otherwise silent endogenous nucleicacid.

In preferred embodiments of the present invention, the resultingrecombinant cells may be delivered to a patient by various methods knownwithin the art including, but not limited to, injection of epithelialcells (e.g., subcutaneously), application of recombinant skin cells as askin graft onto the patient, and intravenous injection of recombinantblood cells (e.g., hematopoietic stem or progenitor cells) or livercells. The total amount of cells that are envisioned for use depend uponthe desired effect, patient state, and the like, and may be determinedby one skilled within the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and may bexenogeneic, heterogeneic, syngeneic, or autogeneic. Cell types include,but are not limited to, differentiated cells such as epithelial cells,endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytesand blood cells, or various stem or progenitor cells, in particularembryonic heart muscle cells, liver stem cells (International PatentPublication WO 94/08598), neural stem cells (Stemple and Anderson, 1992,Cell 71: 973-985), hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, and the like. In a preferred embodiment, the cells utilized forgene therapy are autologous to the patient.

Pharmaceutical Compositions

The compounds, e.g. PDX polypeptides, nucleic acid endowing PDXpolypetides, or a nucleic acid that increases expression of a nucleicacid that encodes ad PDX polypeptide. (also referred to herein as“active compounds”) of the invention, and derivatives, fragments,analogs and homologs thereof, can be incorporated into pharmaceuticalcompositions suitable for administration. Such compositions typicallycomprise the nucleic acid molecule, or protein, and a pharmaceuticallyacceptable carrier. As used herein, “pharmaceutically acceptablecarrier” is intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. Suitable carriers are described in the most recentedition of Remington's Pharmaceutical Sciences, a standard referencetext in the field, which is incorporated herein by reference. Preferredexamples of such carriers or diluents include, but are not limited to,water, saline, finger's solutions, dextrose solution, and 5% human serumalbumin. Liposomes and non-aqueous vehicles such as fixed oils may alsobe used. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe compositions is contemplated. Supplementary active compounds canalso be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a PDX polypeptide or PDX encoding nucleic acid) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811, incorporated fully herein by reference.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by any of a number of routes, e.g., as described in U.S.Pat. No. 5,703,055. Delivery can thus also include, e.g., intravenous ininjection, local administration (see U.S. Pat. No. 5,328,470) orstereotactic injection (see e.g., Chen el al. (1994) PNAS 91:3054-3057).The pharmaceutical preparation of the gene therapy vector can includethe gene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells that producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

SPECIFIC EXAMPLES Example 1 Recombinant Adenoviruses

AdCMVPDX-1 was constructed as described in by R. Seijffers et al.Endocrinology 140:1 133(1999). It contains the STF-1 CDNA, the rathomolog of PDX-1 ligated into BamH1 site of pACCMVpLpA vector.

AdCMVβ-gal, contains the nuclear localization signal forβ-galactosidase.

AdCMV-hIns, contains the human insulin CDNA under the control of theheterologous cytomegalovirus promoter.

AdRIP-1-hIns, contains the human insulin cDNA under the control of therat insulin n promoter-1 (RIP-1). RIP-1, is 410 bases of the 5′ flankingDNA region of the rat insulin-1 gene.

Example 2 Determination of PDX-1 Induced Endogenous Insulin GeneExpression and Activation of Ectopically Co-delivered Insulin Promoter

To assess the effect of ectopic PDX-1 expression in the liver, maleBalb/c and C57BL/6 mice (11-14 week old ) were injected with 2×10⁹plaque forming units (in 0.2 ml saline) of AdCMV-PDX-1 recombinantadenovirus into the tail vein. As controls, mice were similarityinjected with AdCMV-β-gal, or AdCMV-hIns and AdRlP-1-hIns recombinantadenoviruses. The animals were housed in an air-conditioned environment,under a 12-hour light/dark cycle, on a regular unrestricted diet, andsacrificed one week following virus administration. The liver, spleen,pancreas and kidney were dissected and were immediately frozen in liquidnitrogen, and stored at −70° C. for total RNA isolation.

PDX-1 and insulin gene expression was determined by RT-PCR Total RNA wasisolated from frozen tissues using RNAzol (CINNA-BIOTEX). RNA sampleswere treated by 10 ul of DNase I (Promega). CDNA was prepared by reversetranscription, using 1 μg DNA-free total RNA and 0.5 μg oligo(dT)₁₅ 1.5μl of RT reaction was amplified using primers and PCR conditions asindicated in Table 1 below. PCR was carried out in a GeneAmp PCR system2400 (Perkin Elmer), and products were separated on 1.7% agarose gel. Aseparate PCR reaction was carried out for each RNA sample withoutreverse transcriptase, to ensure that the amplified product was not dueto DNA contamination. The primers were designed to detect the only theectopic rat PDX-1 expression not the mouse homolog. The primers for mI-2amplification are located on different exons. The first step of sampledenaturation was identical for all amplified genes: 94° C. for 1 minute.

Analysis of the total RNA revealed that AdCMV-PDX-1 administrationresulted in PDX-1 expression mainly in liver. Spleen, pancreas andkidney from the same mice tested negative by RT-PCR for the rat homologof PDX-1.

75% (25 of 35) of the mice that tested positive for the ectopic ratPDX-1 message expressed the mI-2 gene whereas 35% of the mices expressedmI-1 gene (FIG. 1). To determine whether this disparity of expressionbetween mI-2 and mI-1 was due the mI-1 promotor being differentiallyeffected by the identity or the levels of transcription factors presentin PDX-1 expressing liver cells, AdRIP-1-hIns recombinant adenovirus wasco-delivered with AdCMV-PDX-1 to mice as described above. Asdemonstrated in FIG. 1, in livers where PDX-1 induced only theexpression of the endogenous mI-2, it also activated the rate insulin-1promoter (RIP-1). This suggests that the different levels of DNAmethylation or distinct chromatin structure could be the cause of thelow efficiency of the activation of the endogenous mI-1 expression byPDX-1 expression in the liver. Furthermore these data demonstrate thecapacity to activate the β-cell specific insulin promotor in liver whenco-delivered with PDX.

The expression of the endogenous mouse insulin and the ectopic humaninsulin genes was not induced by treatment with the same concentrationof the control recombinant adenoviruses AdCMV-β-gal, or AdCMV-hIns andAdRIP-1hIns, respectively (n=20). These results demonstrate that PDX-1is essential and sufficient to induce expression of the endogenousinsulin genes and to activate RIP-1 in an extra-pancreatic tissue.

Example 3 Determination of PDX-1 Induced Somatostatin Gene Expressionand Protein Production In-vivo

Animals were treated with recombinant adenovirus as described in EXAMPLE2. Somatostatin gene expression was determined by RT-PCR as described inEXAMPLE 2, according to the conditions described in Table 1.

As demonstrated in FIG. 3 livers in mice treated with AdCMV-PDX-1exhibited somatostatin gene expression. Mice treated with AdCMV-PDX-1exhibited positive staining for the somatostatin prolin in liver tissueanalyzed by inmmunochemistry. Mice treated with AdCMV-β-gal did notexpress somatostatin.

Example 4 Determination of PDX-1 Induced Glucagon Gene Expression

Animals were treated with AdCMVPDX-1 recombinant adenovirus as describedin EXAMPLE 2 Glucagon gene expression was determined by RT-PCR asdescribed in EXAMPLE 2, using conditions and primers as described inTable 1.

As demonstrated in FIG. 3 livers in mice treated with AdCMV-PDX-1exhibited glucagon gene expression. Mice treated with AdCAdV-β-gal didnot express glucagon.

Example 5 Determination of Prohormone Convertase 1/3 Induced GeneExpression

Animals were treated with recombinant adenovirus as described in EXAMPLE2. Prohormone convertase 1/3 (PC1/3) gene expression was determined byRT-PCR as described in EXAMPLE 2 with the exception that cDNA wasreverse-transcribed using a gene specific oligonucleotide(TCCAGGTGCCTACAG GATTCTCT) (SEQ ID NO: 1) instead of oligo (dT)₁₅). Asdemonstrated in FIG. 3 only livers from animals treated with PDX-1exhibited the induction of PC1/3 expression, a member of the Kexinfamily proteases, PC1/3 expression is restricted to endocrine andneuroendocrine cells with regulated secretory pathway. Taken togetherwith the capacity to retain the produced hormones in intracellularcompartments suggests a PDX-1 dependent induction of an endocrinephenotype which includes the induction of a regulated pathway forhormone production, processing, storage and secretion.

TABLE 1 RT-PCR analysis for determination of PDX-1 inducedgene-expression. PCR Conditions Primer Sequences Annealing ExtentionGene 5′-3′ PCR Product ° C. sec ° C. sec Cycles Rat PDX-1 F:CCAGTTTGCAGGCTCGCTGG (SEQ ID NO: 2) 279 bp 62 60 72 60 31 (ectopic) R:GCTGCGTATGCACCTCCTGC (SEQ ID NO: 3) Human Insulin F:CTTTGTGAACCAACACCTGTGC (SEQ ID NO: 4) 239 bp 63 60 72 60 38 (ectopic) R:GCAGATGCTGGTACAGCATTGT (SEQ ID NO: 5) Mouse Insulin I F:TTGCCCTCTGGGAGCCCAAA (SEQ ID NO: 6) 253 bp 62 60 72 60 38 R:CAGATGCTGGTGCAGCACTG (SEQ ID NO: 7) Mouse Insulin II F:TCTTCCTCTGGGAGTCCCAC (SEQ ID NO: 8) 259 bp 62 60 72 60 38 R:CAGATGCTGGTGCAGCACTG (SEQ ID NO: 9) Mouse-actin F: ATGGATGACGATATCGCT(SEQ ID NO: 10) 500 bp 56 45 72 60 35 R: ATGAGGTAGTCTGTCAGGT (SEQ ID NO:11) Mouse PC1/3 F: CTGGTTGTCTGGACCTCTGAGTA (SEQ ID NO: 12) 361 bp 55 4572 60 38 R: CCAACAGCAGAAGTGAGTGTGAC (SEQ ID NO: 13) Mouse PDX-1 F:CAAGCTCGCTGGGATCACTGGAGCAG (SEQ ID NO: 14) 421 bp 58 45 72 60 38(endogenous) R: GATGTGTCTCTCGGTCAAGTTCAACATC (SEQ ID NO: 15) Mouse & RatF: CCTGGCTTTGGGCGGTGTCA (SEQ ID NO: 16) 165 bp 68 45 72 60 38somatostatin R: CTCGGGCTCCAGGGCATCATTC (SEQ ID NO: 17) Mouse glucagon F:ACCAGCGACTACAGCAAATACCTC (SEQ ID NO: 18) 242 bp 60 45 72 60 38 R:AGCAATGGCGACTTCTTCTGG (SEQ ID NO: 19) rat insulin-1 F:GTGACCAGCTACAATCATAG (SEQ ID NO: 20) 370 bp 57 45 72 60 38 R:AGTTCTCCAGTTGGTAGAGG (SEQ ID NO: 21) Rat-actin F: CGTAAAGACCTCTATGCCAA(SEQ ID NO: 22) 350 bp 57 45 72 60 35 R: AGCCATGCCAAATGTGTCAT (SEQ IDNO: 23)

Example 6 PDX-1 Induced Proinsulin Synthesis in Livers

Animals were treated with recombinant adenovirus as described in EXAMPLE2. Liver, spleen, pancreas and kidney were dissected. Portions of thetissue fixed in 4% formaldehyde and embedded in paraffin forimmunohistochemical staining. The remaining liver and pancreatic tissueswere homogenized in 70% ethanol-0.18N HCl, lyophilized and resuspendedin PBS (phosphate buffered saline) for RIA determination of IRI content.

Immunohistochemisty

Five μm sections of paraffin-embedded tissues were deparaffinized,incubated in 3% H₂O₂, and then, either microwaved in citrate buffer forantigen retrieval prior to incubation in blocking solution (PDX-1detection), or immediately exposed to the blocking solution (insulindetection). (Histomouse™-SP Kit, Zymed laboratories, Calif., USA).

PDX-1 detection: sections were incubated overnight at 4° C. withantiserum raised against the N-terminal portion of frog PDX-1.

Insulin detection: sections were incubated for 1 hour at 37° C. with amonoclonal anti-human-insulin (Sigma, St.-Louis Mo.).

Slides were exposed to the secondary biotinylated IgG for 30 minutes,incubated in streptavidin-peroxidase followed by chromogen-peroxidesolution.

Immunohistochemical analysis of liver sections from mice treated withPDX-1, revealed expression of the homeoprotein in 30-60% of hepatocytenuclei, with 0.1-1% of the liver cells staining positive for(pro)insulin. Control AdCMVβ-gal treated livers, did not stain positivefor (pro)insulin although β-galactosidase activity was evident in 50% ofthe nuclei. Livers from mice treated by AdCMV-hIns, did not stainpositive for insulin in the hepatic sections, although serum IRI fromthe same mice was three fold increased, as were serum IRI levels inPDX-1 treated mice. The fact that the ectopic expression of PDX-1 butnot of insulin resulted in positive immunostaining for (pro)insulin maysuggest the induction of a cellular modulation which supports insulinretention in a small subpopulation of liver cells, (secretory vesicleswhich belong to the regulated pathway, characteristic to endocrinecells, but not to liver cells), which may have shifted toward a β-cellphenotype.

Radioimmunoassay

To determine whether hepatic insulin mRNA is effectively translated intoprotein, immunoreactive insulin (IRI) content was tested in extractsderived from hepatic tissue by radioimmunoassay (RIA). Livers from PDX-1treated mice that tested positive for insulin gene expression by RT-PCR(FIG. 1) contained about 25 fold more IRI than livers of animals treatedby a control virus (Table 2). Mean IRI levels in extracts derived fromPDX-1 treated livers was 20.7±3.97 μU/mg protein, while in controllivers, IRI was only 0.78±0.25 μU/mg protein. The background level ofinsulin measured in control liver samples possibly represents insulin(of pancreatic origin) bound to its receptors which are abundant in thisorgan. While IRI detected in PDX-1 treated liver extracts was <1% of thelevels detected in pancreatic extracts (Table 2), serum IRI levels inPDX-1 treated mice were almost 3-fold higher compared to controls(323±48.4 vs. 118.2±23.7 μU/ml, respectively (Table 2)), indicating thatinsulin was being synthesized and a large portion of it secreted intothe blood stream. These data indicate that the insulin gene expressioninduced by the molecular manipulation is successfully translated intospecific hepatic production of the pro/hormone.

Immunoreactive insulin detected in PDX-1 treated livers was less than 1%of IRI levels in pancreatic extracts (Table 2). The IRI valuesdetermined by radioimmunoassay (RIA) in liver extracts mayunder-estimate the actual insulin production in this organ. The antibodywe used for RIA preferentially binds the processed hormone, and has only60% cross-reactivity with proinsulin, which is expected to be presentmainly in hepatocytes and to a much lower extent in pancreas.

Example 7 Blood Glucose and Serum Insulin Levels

Animals were treated with recombinant adenovirus as described in EXAMPLE2. Prior to sacrifice, blood was drawn from the inferior vena cava fordetermination of glucose concentration (Accutrend® GC, BoehringerMannheim, Mannheim, Germany) and insulin levels by radioimmunoassay(Coat-a-count, DPC, Los-Angeles, Calif., USA, using rat insulinstandards, (Linco), the anti-insulin antibody used has only 60%cross-reactivity with human proinsulin).

Mice treated by AdCMV-PDX-1 recombinant adenoviruses were nothypoglycemic, however, their blood glucose levels were significantlylower than of mice treated by AdCMV-β-gal or AdCMV-Luc [197±11.2 vs.228±15.74 mg/dl, respectively (Table 2). Plasma immunoreactive insulinlevels were significantly higher in PDX-1 treated mice compared tocontrols (323±48.4 vs. 118.2±23.7 μU/ml respectively (Table 2).

The three fold increase in serum IRI levels in PDX-1 treated mice,cannot by itself explain the twenty-fivefold increase (Table 2) inhepatic IRI content demonstrated in PDX-1 treated liver extracts. Thus,the increase in hepatic pro/insulin content originates from localproduction.

TABLE 2 Blood glucose and immunoreactive insulin (IRI) levels in serumand liver extracts. Control virus AdCMV-PDX-1 treated mice treated miceBlood glucose, mg/dl  228 ± 15.74 (n = 18) 197 ± 11.2 (n = 40) SerumIRI, μU/ml 118.2 ± 23.7 (n = 14) 323 ± 48.4 (n = 26) Liver extracts IRIμU/mg  0.78 ± 0.25 (n = 10) 20.7 ± 3.97 (n = 12)  protein Pancreasextracts IRI   2627 ± 24 (n = 6) μU/mg protein

Example 8 HPLC Analysis of Insulin-related Peptides

Animals were treated with recombinant adenovirus as described in EXAMPLE2. Liver, and pancreas were dissected and homogenized in 70%ethanol-0.18N HCl, lyophilized and resuspended in 0.1 M HCl—0.1% BSA forHPLC analysis.

Insulin-related peptides from the liver and pancreatic extracts wereresolved by reverse-phase HPLC using Lichrospher 100 RP-18 column(Merck, Darmstadt, Germany) and elution conditions as described by Grosset al. One ma fractions were collected into tubes containing 0.1 ml 0.1%BSA in water, dried in a Speed-Vac apparatus and reconstituted in 1 mlRIA buffer (0.1% BSA in PBS) for peptide determination by RIA. Guineapig antiporcine insulin antibodies (Linco, St Charles, Mo.) with eitherrat or human insulin standards were used for determination of mouse orhuman IRI, respectively.

HPLC analysis of hepatic IRI content from PDX-1 treated mice revealed59±7% (n=3) conversion into filly processed mI-1 and mI-2. Incomparison, pancreatic extracts contained 85±5% (n=3) mature insulin(FIG. 2) Whereas, ectopic expression of human insulin (AdCMV-hIns) didnot result in retention of IRI in the liver cells except for one liverin which most of the extracted IRI was immature insulin. This is in linewith previous observations in transfected FAO cells in which noretention of the insulin gene product observed and most of it wassecreted by the constitutive secretory pathway. These data demonstratesthat ectopic PDX-1 expression in liver induces a cellular machinary,characteristic to endocrine tissue capable of processing the inducedprohormone, and is not induced when only proinsulin is ectopicallyexpressed in liver. Thus, inducing an extended β-cell phenotype in livercells by ectopic PDX-1 expression.

Example 9 Biological Activity of Hepatic Pro/insulin Production

The ability of PDX-1-induced hepatic insulin production to control bloodglucose levels in diabetic mice was studied. C57BL/6 mice were rendereddiabetic (>600 mg/dl) with ketoacidosis, 24 hours after 200 mg/kgintraperitoneal STZ injection. 24-48 hours after STZ injection, micewere treated by either AdCMV-PDX-1 or by AdCMVβ-gal (control)recombinant adenoviruses administered via the tail vein, in salinesolution. As demonstrated in FIG. 4, AdCMV-PDX-1 treated mice, exhibitedgradual decrase in blood glucose levels from about 600 to 200-300 mg/dlstarting two days after recombinant adenoviral treatment In contrast, inthe control AdCMVβ-gal treated mice, hyperglycemia persisted and wasaccompanied by increased rate of mortality, 12 out of 22 tested died,with severe ketoacidosis 1-3 days after adenovirus treatment.Furthermore, both groups lost weight after induction of hyperglycemia,and did not regain it back before mice were sacrificed. In summary, thedata demonstrate that expression of PDX-1 is sufficient to inducemature, biologically active insulin production in liver whichameliorates hyperglycemia in mice bearing ablated 0cell function.

Example 10 In-vitro Activation of Insulin Promoter by Ectopic PDX-1Expression

PDX-1 activates rat insulin-1 promotor when co-delivered with arecombinant adenovirus AdRip-1hIns in which human insulin expression isdelivered by a rat insulin-1 promoter. (See, EXAMPLE 2 and FIG. 1. PDX-1was shown to be sufficient to activate rat insulin promoter-1 in-vitroin rat liver cells. Primary cultures if mature and fetal hepatocyteswere cultured on collagen-1 covered tissue culture dishes in serum freechemically defined media. Two days after to plating cells were treatedby either AdCMV-PDX-1& AdRIP-1hIns or by AdCMV β-gal & AdRIP-1hIns. 48hours after adenoviral treatment, total RNA was extracted and proinsulingenes expression was assessed as described in EXAMPLE 2.

PDX-1 activated the ectopically expressed RIP-hIns (rat insulinpromoter-1, 410 bps of this promoter, driving human insulin, introducedvia recombinant adenovirus), while β-gal did not possess such acapacity. ( FIG. 5)

Example 11 In-vitro Induction of Endogenous Somatostatin Gene Expressionin Hepatocytes

Primary cultures of hepatocytes isolated from fetal (E14-Fisher-344rats) were cultured and treated by recombinant adenoviruses as describedin EXAMPLE 9. Somatostatin gene expression was detected in reversetranscribed total RNA samples as described in EXAMPLE 2, using primersand RT-PCR conditions as described in Table 1.

The data demonstrate that ectopic PDX-1 expression in hepatocytesin-vitro induces the expression of the endogenous, otherwise silentsomatostatin gene expression in hepatocytes, in-vitro (FIG. 6).

Example 12 In-vitro Induction of Endogenous Insulin Gene Expression inHepatocytes

Primary cultures of fetal (E14-Fisher-344 rats) were cultured andtreated by recombinant adenoviruses as described in EXAMPLE 10. Ratinsulin 1 gene expression was detected in reverse transcribed total RNAsamples as described in EXAMPLE 2, using primers and RT-PCR conditionsas described in Table 1.

The data demonstrate that ectopic PDX-1 expression in primary culture offetal hepatocytes in-vitro induces the expression of the endogenous,otherwise silent insulin gene expression (FIG. 6).

Example 13 Ectopic PDX-1 Expression in Liver Cells Induces anIntracellular Compartment Characteristic of Endocrine and NeuroendocrineCells Which Allows the Retention of the Produced Hormones, and itsRegulated Secretion

Mice were treated with either Ad-CMVhIns or AdCMVPDX-1 as described inEXAMPLE 2. Treatment resulted in a three-fold increase serum IRIdemonstrating human insulin production by liver cells (FIG. 1). Cellspositive for the insulin protein by immnunocytochemistry were detectedonly in AdCMVPDX-treatment. Moreover, HPLC analysis of liver extractsdetected only trace levels of IRI in liver extracts all of itunprocessed in the Ad-CMVhIns treated mice compared to 25 fold increasein the AdCMVPDX-1 treated mice. Furthermore, 59% of the insulin producedin AdCMVPDX-1 treated mice was processed. In addition, only liverstreated by AdCMVPDX-1 exhibited the induction of the prohormoneprocessing enzyme PC1/3 which is characteristic only to cells capable ofregulated pathway for insulin processing storage and regulatedsecretion. These data demonstrate that PDX induces the regulatedsecretion of insulin in liver cells

Example 14 Identification of Nucleic Acids the Expression of Which isModulated by PDX

Nucleic acids modulated by PDX are identified by ectopic PDX expression.Nucleic acids that are not expressed in control treated extra-pancreaticislet tissue, as compared to pancreatic tissue are the nucleic acidsmodulated by PDX. These nucleic acids so identified are used astherapeutic compounds to treat pancreatic associated disorders.

Identification of the target genes is performed by either subtractivelibraries, commercially available microarray Chips (Incyte, orAffimetrix), or membrane hybridizations (CLONTECH. Atlas™ expressionarrays, or Multiple Tissue Northern (MTN) Blots). RNA isolation fromtreated tissues, its purification, and cDNA probe synthesis is performedaccording to manufacturer instructions.

The genes which are expressed in the PDX treated non-pancreatic islettissue and are also present in pancreatic islets probed membranes orchips, but not in control treated non-pancreatic islet tissue, are thedirect and non-direct PDX target genes, which represent the islet cellscharacteristic profile of gene expression. Discrimination between director indirect is elucidated by candidate target gene promoter analysis byelectromobility shift assay (EMSA) as in FIG. 7, At and promoterfootprinting (as described in Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

Example 15 Inducing Regulated Expression of a Desired EctopicallyExpressed Gene in Host Tissue

This EXAMPLE illustrates the induction of regulated expression of anyreporter, in addition to insulin. When PDX activates the insulinpromoter in non pancreatic islet tissue, and mediates its glucose andgrowth factors sensing ability, than, any additional promotor will besimilarly regulated by glucose and growth factors. Thus, this inventioncan be utilized to nutritionally and hormonally regulate expression ofnumerous secreted/or non secreted factors such as for example, glucagon,growth hormone, steroid hormones which are driven by the insulinpromoter thus controlling their transcription, and regulated secretion,from an otherwise non-endocrine tissue. (FIG. 7.)

Example 16 Identification of PDX Location in the Hierarchy of B or IsletCell Specific Transcription Factors

This EXAMPLE illustrates the identification of the PDX location in thehierarchy of β-cell or islet cell specific transcription factors. Everytranscription factor expressed in pancreatic islets but is not inducedby ectopic PDX-1 expression in liver, could cooperate with PDX for theinduction of a more comprehensive, complete or close to complete β-cellphenotype in non-endocrine-pancreatic tissue, such as liver. Thedetection of induced expression of islet cell specific transcriptionfactors in liver is performed as in EXAMPLE 2, using the appropriateprimers and conditions the example of which is elaborated in Table 1.

An additional method to analyze the activity of transcription factors isperformed by footprinting, and by

ElectroMobility Shift Assays (EMSA): Nuclear extracts (3-4 μg ofprotein) were incubated on ice for 10 minutes in DNA binding mixturecontaining 10% Glycerol, 15 mM Hepes (pH 7.9), 150 mM KCl, 5 mM DTT and0.3 μg of poly dIdC, poly dAdT (SIGMA St-Louis Mo.). After the firstincubation, approximately 0.2 ng of the probe was added for anadditional 25 minutes incubation on ice, The binding reaction wasanalyzed on a native 4% polyacrylamide gel.

Oligonucleolides (probes). Synthetic double-stranded oligonucleotidesare end-labeled with [a32P]ATP using the Klenow fragment of DNApolymerase. The sequences of oligonucleotides A3/A4 which is an examplefor PDX-1 binding site (one of them) on the insulin promoter 5′GATCTGCCCCTTGTTAATAATCTAATG 3′(SEQ ID NO: 24). The sequence for A1 (additionalPDX-1 binding site on insulin promoter) is 5′GATCCGCCCTTAATGGGCCAAACGGCA-3′ (SEQ ID NO: 25). The labeled oligos areused as probes for electromobility shift assays, as described in FIG. 7.The identity of PDX-1 is double estimated by supershift using a specificantibody which prevents the PDX-1 binding to its cognate locus on thepromoter, or that increases the molecular weight of the complexseparated on PAGE (antibody+pdx-1+probe) compared to that which includesonly pdx-1+labeled probe (last two lanes in FIG. 7).

Equivalents

From the foregoing detailed description of the specific embodiments ofthe invention, it should be apparent that unique methods of inducingpancreatic hormone production has been described. Although particularembodiments have been disclosed herein in detail, this has been done byway of example for purposes of illustration only, and is not intended tobe limiting with respect to the scope of the appended claims whichfollow. In particular, it is contemplated by the inventor that varioussubstitutions, alterations, and modifications may be made to theinvention without departing from the spirit and scope of the inventionas defined by the claims.

25 1 23 DNA Artificial Sequence Description of Artificial Sequencechemically synthesized 1 tccaggtgcc tacaggattc tct 23 2 20 DNAArtificial Sequence chemically synthesized 2 ccagtttgca ggctcgctgg 20 320 DNA Artificial Sequence chemically synthesized 3 gctgcgtatgcacctcctgc 20 4 22 DNA Artificial Sequence chemically synthesized 4ctttgtgaac caacacctgt gc 22 5 22 DNA Artificial Sequence chemicallysynthesized 5 gcagatgctg gtacagcatt gt 22 6 20 DNA Artificial Sequencechemically synthesized 6 ttgccctctg ggagcccaaa 20 7 20 DNA ArtificialSequence chemically synthesized 7 cagatgctgg tgcagcactg 20 8 20 DNAArtificial Sequence chemically synthesized 8 tcttcctctg ggagtcccac 20 920 DNA Artificial Sequence chemically synthesized 9 cagatgctggtgcagcactg 20 10 18 DNA Artificial Sequence chemically synthesized 10atggatgacg atatcgct 18 11 19 DNA Artificial Sequence chemicallysynthesized 11 atgaggtagt ctgtcaggt 19 12 23 DNA Artificial Sequencechemically synthesized 12 ctggttgtct ggacctctga gta 23 13 23 DNAArtificial Sequence chemically synthesized 13 ccaacagcag aagtgagtgt gac23 14 26 DNA Artificial Sequence chemically synthesized 14 caagctcgctgggatcactg gagcag 26 15 28 DNA Artificial Sequence chemicallysynthesized 15 gatgtgtctc tcggtcaagt tcaacatc 28 16 20 DNA ArtificialSequence chemically synthesized 16 cctggctttg ggcggtgtca 20 17 22 DNAArtificial Sequence chemically synthesized 17 ctcgggctcc agggcatcat tc22 18 24 DNA Artificial Sequence chemically synthesized 18 accagcgactacagcaaata cctc 24 19 21 DNA Artificial Sequence chemically synthesized19 agcaatggcg acttcttctg g 21 20 20 DNA Artificial Sequence chemicallysynthesized 20 gtgaccagct acaatcatag 20 21 20 DNA Artificial Sequencechemically synthesized 21 agttctccag ttggtagagg 20 22 20 DNA ArtificialSequence chemically synthesized 22 cgtaaagacc tctatgccaa 20 23 20 DNAArtificial Sequence chemically synthesized 23 agccatgcca aatgtgtcat 2024 27 DNA Artificial Sequence chemically synthesized 24 gatctgccccttgttaataa tctaatg 27 25 27 DNA Artificial Sequence chemicallysynthesized 25 gatccgccct taatgggcca aacggca 27

What is claimed is:
 1. A method of inducing pancreatic hormoneexpression in the liver of a mammal, wherein said pancreatic hormone isselected from the group consisting of insulin, somatostatin, andglucagon, said method comprising administering to a mammal an adenoviralparticle comprising an adenovirus vector comprising a cytomegalovirus(CMV) promoter operably linked to a nucleic acid encoding a pancreaticand duodenal homobox 1 (PDX-1) polypeptide in an amount sufficient toinduce said pancreatic hormone expression in said liver in said mammal.2. The method of claim 1, wherein administering said vector increaseshepatic insulin levels in said mammal.
 3. The method of claim 1, whereinadministering said vector increases serum insulin levels in said mammal.4. The method of claim 1, wherein the mammal is a rodent or human. 5.The method of claim 1, wherein the mammal is further administered atransfection agent.
 6. The method of claim 1, wherein the administeringis by intravenous delivery.
 7. A method of inducing a pancreatic isletgene expression profile in a liver cell of a subject, said methodcomprising administering to a subject an adenoviral particle comprisingan adenovirus vector comprising a cytomegalovirus (CMV) promoteroperably linked to a nucleic acid encoding a pancreatic and duodenalhomobox 1 (PDX-1) polypeptide in an amount sufficient to induce saidpancreatic islet gene expression in said liver cell in said subject. 8.A method of inducing insulin expression in the liver of a mammal, saidmethod comprising administering to a mammal an adenoviral particlecomprising an adenovirus vector comprising a cytomegaloviras (CMV)promoter operably linked to a nucleic acid encoding a pancreatic andduodenal homobox 1 (PDX-1) polypeptide in an amount sufficient to inducesaid insulin expression in said liver of said mammal.
 9. A method ofinducing glucagon expression in the liver of a mammal, said methodcomprising administering to a mammal an adenoviral particle comprisingan adenovirus vector comprising a cytomegalovirus (CMV) promoteroperably linked to a nucleic acid encoding a pancreatic and duodenalhomobox 1 (PDX-1) polypeptide in an amount sufficient to induce saidglucagon expression in said liver of said mammal.
 10. A method ofinducing somatostatin expression in the liver of a mammal, said methodcomprising administering to a mammal an adenoviral particlecomprising anadenovirus vector comprising a cytomegalovirus (CMV) promoter operablylinked to a nucleic acid encoding a pancreatic and duodenal homobox 1(PDX-1) polypeptide in an amount sufficient to induce said somatostatinexpression in said liver of said mammal.
 11. A method of inducingprohormone convertase 1/3 (PC1/3) expression in the liver of a mammal,said method comprising administering to a mammal an adenoviral particlecomprising an adenovirus vector comprising a cytomegalovirus (CMV)promoter operably linked to a nucleic acid encoding a pancreatic andduodenal homobox 1 (PDX-1) polypeptide in an amount sufficient to inducesaid PC1/3 expression in said liver of said mammal.
 12. A method ofinducing pancreatic hormone expression in a liver cell, wherein saidpancreatic hormone is selected from the group consisting of insulin,somatostatin, and glucagon, said method comprising contacting said cellwith an adenovirus vector comprising a cytomegalovirus (CMV) promoteroperably linked to a nucleic acid encoding a pancreatic and duodenalhomobox 1 (PDX-1) polypeptide, thereby inducing said pancreatic hormoneexpression in said liver cell.
 13. A method of inducing insulinexpression in a liver cell, said method comprising contacting said cellwith an adenovirus vector comprising a cytomegalovirus (CMV) promoteroperably linked to a nucleic acid encoding a pancreatic and duodenalhomobox 1 (PDX-1) polypeptide, thereby inducing said insulin expressionin said liver cell.
 14. A method of inducing somatostatin expression ina liver cell, said method comprising contacting said cell with anadenovirus vector comprising a cytomegalovirus (CMV) promoter operablylinked to a nucleic acid encoding a pancreatic and duodenal homobox 1(PDX-1) polypeptide, thereby inducing said somatostatin expression insaid liver cell.
 15. A method of inducing glucagon expression in a livercell, said method comprising contacting said cell with an adenovirusvector comprising a cytomegalovirus (CMV) promoter operably linked to anucleic acid encoding a pancreatic and duodenal homobox 1 (PDX-1)polypeptide, thereby inducing said glucagon expression in said livercell.
 16. A method of inducing prohormone convertase 1/3 (PC1/3)expression in a liver cell, said method comprising contacting said cellwith an adenovirus vector comprising a cytomegalovirus (CMV) promoteroperably linked to a nucleic acid encoding a pancreatic and duodenalhomobox 1 (PDX-1) polypeptide, thereby inducing said PC1/3 expression insaid liver cell.
 17. A composition comprising an adenovirus vectorcomprising a cytomegalovirus (CMV) promoter operably linked to a nucleicacid encoding a pancreatic and duodenal homobox 1 (PDX-1) polypeptide,and a carrier.